CD44 is an adhesion molecule expressed in cancer stem-like cells. Here, we show that a CD44 variant (CD44v) interacts with xCT, a glutamate-cystine transporter, and controls the intracellular level of reduced glutathione (GSH). Human gastrointestinal cancer cells with a high level of CD44 expression showed an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS). Ablation of CD44 induced loss of xCT from the cell surface and suppressed tumor growth in a transgenic mouse model of gastric cancer. It also induced activation of p38(MAPK), a downstream target of ROS, and expression of the gene for the cell cycle inhibitor p21(CIP1/WAF1). These findings establish a function for CD44v in regulation of ROS defense and tumor growth.

This paper derives a speech parameter generation algorithm for HMM-based speech synthesis, in which speech parameter sequence is generated from HMMs whose observation vector consists of spectral parameter vector and its dynamic feature vectors. In the algorithm, we assume that the state sequence (state and mixture sequence for the multi-mixture case) or a part of the state sequence is unobservable (i.e., hidden or latent). As a result, the algorithm iterates the forward-backward algorithm and the parameter generation algorithm for the case where state sequence is given. Experimental results show that by using the algorithm, we can reproduce clear formant structure from multi-mixture HMMs as compared with that produced from single-mixture HMMs.

In cancer metastasis, various environmental stressors attack the disseminating cells. The successful colonization of cancer cells in secondary sites therefore requires the ability of the cells to avoid the consequences of such exposure to the stressors. Here we show that orthotopic transplantation of a CD44 variant isoform-expressing (CD44v + ) subpopulation of 4T1 breast cancer cells, but not that of a CD44v − subpopulation, in mice results in efficient lung metastasis accompanied by expansion of stem-like cancer cells. such metastasis is dependent on the activity of the cystine transporter xCT, and the stability of this protein is controlled by CD44v. We find that epithelial splicing regulatory protein 1 regulates the expression of CD44v, and knockdown of epithelial splicing regulatory protein 1 in CD44v + cells results in an isoform switch from CD44v to CD44 standard (CD44s), leading to reduced cell surface expression of xCT and suppression of lung colonization. The epithelial splicing regulatory protein 1-CD44v-xCT axis is thus a potential therapeutic target for the prevention of metastasis.

A large number of structurally diverse compounds act as openchannel blockers of NMDA receptors. They may share discrete or overlapping binding sites within the channel. In this study, the effects of mutations in and around the membrane-spanning and pore-forming regions of NMDA receptor subunits were studied with three blockers, MK-801, memantine, and TB-3-4, using recombinant NMDA receptors expressed in Xenopus laevis oocytes. Mutations at the critical asparagine residues in the M2 loop of NR1 and NR2B and at a tryptophan residue in M2 of NR2B reduced block by MK-801, memantine, and TB-3-4. Mutations at residues in the pre-M1, M1, M3, post-M3, and post-M4 regions had differential effects on the three blockers. Many mutations in these regions reduced block by MK-801 and TB-3-4 but had no effect on block by memantine. The differential effects on block by memantine and MK-801 are unlikely to be caused by differences in the size of these blockers. Benzyl rings in MK-801 and TB-3-4 may make hydrophobic interactions with aromatic and hydrophobic amino acid residues in the pore. Some mutations in the pre-M1 and M3 regions generated constitutively open channels, characterized by large holding currents. The effects of the various mutants are discussed in the context of models based on the known structure of the pore of the KcsA potassium channel and on previous studies dealing with solvent accessible residues in NMDA receptor subunits as determined by modification after cysteine mutagenesis.

The targeting of antioxidant systems that allow stem-like cancer cells to avoid the adverse consequences of oxidative stress might be expected to improve the efficacy of cancer treatment. Here, we show that head and neck squamous cell carcinoma (HNSCC) cells that express variant isoforms of CD44 (CD44v) rely on the activity of the cystine transporter subunit xCT for control of their redox status. xCT inhibition selectively induces apoptosis in CD44v-expressing tumor cells without affecting CD44v-negative differentiated cells in the same tumor. In contrast to CD44v-expressing undifferentiated cells, CD44v-negative differentiated cells manifest EGF receptor (EGFR) activation and rely on EGFR activity for their survival. Combined treatment with inhibitors of xCTdependent cystine transport and of EGFR resulted in a synergistic reduction of EGFR-expressing HNSCC tumor growth. Thus, xCT-targeted therapy may deplete CD44v-expressing undifferentiated HNSCC cells and concurrently sensitize the remaining differentiating cells to available treatments including EGFR-targeted therapy.

There are complex interactions between spermine, protons, and ifenprodil at N-methyl-D-aspartate receptors. Spermine stimulation may involve relief of proton inhibition, whereas ifenprodil inhibition may involve an increase in proton inhibition. We studied mutations at acidic residues in the NR1 subunit using voltage-clamp recording of NR1/NR2B receptors expressed in Xenopus oocytes. Mutations at residues near the site of the exon-5 insert, including E181 and E185, reduced spermine stimulation and proton inhibition. Mutation NR1(D130N) reduced sensitivity to ifenprodil by more than 500-fold, but had little effect on sensitivity to spermine and pH. Mutations at six other residues in this region of the NR1 subunit reduced the potency and, in some cases, the maximum effect of ifenprodil. These mutants did not affect sensitivity to pH, glutamate, glycine, or other hallmark properties of N-methyl-D-aspartate channels such as Mg2+ block and Ba2+ permeability. Residues in this region presumably form part of the ifenprodil-binding site. To model this region of NR1 we compared the predicted secondary structure of NR1 (residues 19-400) with the known structures of 1,400 proteins. This region of NR1 is most similar to bacterial leucine/isoleucine/valine binding protein, a globular amino acid binding protein containing two lobes, similar to the downstream S1-S2 region of glutamate receptors. We propose that the tertiary structure of NR1(22-375) is similar to leucine/isoleucine/valine binding protein, containing two "regulatory" domains, which we term R1 and R2. This region, which contains the binding sites for spermine and ifenprodil, may influence the downstream S1 and S2 domains that constitute the glycine binding pocket.

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Bloom's syndrome (BS) is a rare genetic disorder and the cells from BS patients show genomic instability and an increased level of sister chromatid exchange (SCE). We generated BLM ±/± and BLM ±/± /RAD54 ±/± DT40 cells from the chicken B-lymphocyte line DT40. The BLM ±/± DT40 cells showed higher sensitivity to methyl methanesulfonate and elevated levels of SCE as expected. The targeted integration frequency was also increased remarkably in BLM ±/± cells. The SCE frequency increase in BLM ±/± cells was considerably reduced and the enhanced targeted integration observed in BLM ±/± cells was almost completely abolished in BLM ±/± /RAD54 ±/± cells, indicating that a large portion of the SCE in BLM ±/± cells occurs via homologous recombination, and homologous recombination events increase with the defect of BLM function. The BLM ±/± /RAD54 ±/± cells showed a slow growth phenotype and an increased incidence of chromosometype breaks/gaps while each single mutant showed relatively small numbers of chromosome-type breaks/ gaps.